A variety of vectors and hosts have been used for the mass production of recombinant proteins. E. coli has been widely used, but has limited usefulness in the production of proteins that need to be glycosylated or have complicated structures. These problems can be overcome using animal cells, yeast cells, transformed animals, transformed plants, and the like.
Yeast is advantageous in the mass production of proteins, but it is well known that yeast glycosylation is different from human glycosylation and is thus highly immunogenic (Hermeling et al., Pharm. Res. 21(6):897-903 (2004)). Transformed animals have not been commercialized owing to difficulty in the care and maintenance of the animals and potential contamination with microbiological pathogens.
The use of animal cell lines is burdened by the high cost of protein production and the time and expense required for cell line establishment, but is most commonly used for recombinant protein production because this system produces a recombinant protein in a form very similar to that seen in human cells and enables stable protein production and maintenance. However, when transformed for recombinant protein production, most animal cell lines exhibit low expression levels and thus have not yet been used to realize high yield production. Gene amplification is a strategy that is routinely used to overcome the problems associated with animal cell expression systems. The two widely used amplification systems are dihydrofolate reductase (DHFR)-based amplification and glutamine synthetase-based amplification, both of which can considerably increase the recombinant protein yield of animal cell lines. Despite its advantage of improving protein production, gene amplification systems have drawbacks in that they require multiple rounds of gene amplification in order to use high concentrations of methotrexate (hereinafter, referred to simply as “MTX”), which are time-consuming, and the long-term subculture of cell lines leads to gene loss and unstable expression.
Many attempts have been made to overcome the drawbacks of gene amplification systems. For example, Korean Pat. Registration No. 0162021 employs a DHFR gene which is placed under the control of a partially deleted SV40 promoter. Korean Pat. Registration No. 0493703 describes the introduction of a mutation into a cytomegalovirus (CMV) promoter so as to alter the affinity of methylated DNA-binding protein to its recognition sequence in the promoter. Korean Pat. Registration No. 0184778 employs the 5′-noncoding region of an immunoglobulin heavy chain binding protein (Bip) as an internal ribosome entry site (IRES) in order to place a DHFR gene under the control not of an independent promoter but of a transcription control sequence of a recombinant protein of interest.
Korean Pat. Registration No. 0162021 was directed to the control of the activity of the SV40 promoter by deleting 128 to 270 nucleotides. However, the role of the deleted sequence is not accurately known, and the role of the remaining sequence is not mentioned. In particular, because this patent does not show that gene amplification is not increased any further at concentrations of MTX higher than 20 nM under a condition not containing a control, it does not provide any support at all for the change in the DHFR promoter activity and the optimized expression at a low concentration of MTX. Korean Pat. Registration No. 0493703 intended to achieve effective gene amplification by modifying the recognition sequence of methylated DNA-binding protein in the CMV promoter, but the amplification effect on expression is lower than that of other methods. In Korean Pat. Registration No. 0184778, the IRES-dependent expression, conducted instead of the use of an independent promoter, allows gene amplification merely at levels of MTX as low as several micromoles (μM), and a roughly 30-fold expression increase. Thus, the amplification of the DHFR gene cannot be predicted from the modification of a general promoter, and the application of a modified promoter will be determined only when gene amplification is substantially performed using various kinds of promoters.
Human erythropoietin (EPO), which is illustrated as an example of recombinant proteins in the present invention, is a glycoprotein of about 34 kDa, but the molecular mass of the peptide chain (non-glycosylated EPO) is only about 18 kDa. EPO is synthesized in the kidney in response to anemia, hypoxia or bleeding, and stimulates the production of red blood cells and maintains homeostasis. EPO is present at about 10 to 20 mIU/mL in adults, and renal dysfunction brings about severe anemia (Jacobson, et al., Nature, 179:633-634 (1957)). Thus, EPO has been used as a therapeutic agent for chronic renal failure and anemia caused by various factors. In the past, EPO was harvested from the blood plasma of animals, or from the blood or urine of patients having aplastic anemia, who produce EPO at higher levels than healthy persons, but EPO is obtained in an unstable form and in low yield. Urinary EPO from healthy persons is obtained at low concentrations, and needs to be highly purified because urine contains an inhibitor of EPO activity (see, U.S. Pat. Nos. 4,397,840, 4,303,650 and 3,865,810). Since it is difficult to obtain large amounts of highly pure EPO from the blood or the urine, EPO preparation methods using genetic recombination techniques have been developed. However, since glycosylation is required for the in vivo activity of EPO, when an EPO gene is cloned and expressed in E. coli or yeast, EPO is not glycosylated in its native form, and thus does not display its biological activity. Hence, the use of a recombinant animal cell line is essentially required for EPO production. In the case of using a recombinant animal cell line, EPO is usually produced based on a DHFR gene amplification system (Malik et al., DNA and Cell Bio., 6:453-459 (1992)).